PROJECT SUMMARY Inhibitory mechanisms within the tumor microenvironment represent major barriers to effective antitumor immunity. Although striking efficacy in a variety of tumor types has been reported in clinical trails with inhibitory receptor blockade, it's clear that additional inhibitory mechanisms will need to be targeted to substantially improve therapeutic outcome in most tumor types. Regulatory T cells (Tregs) are potent inhibitors of anti-tumor immunity. Although targeted Treg ablation results in rapid tumor shrinkage in multiple models of human cancer, this is not a viable therapeutic approach due to the subsequent severe autoimmune and inflammatory consequences. Thus more targeted approaches to limit Treg activity selectively in the tumor microenvironment are needed, along with in depth understanding of their mechanism of action. Although Tregs use multiple inhibitory mechanisms to control T cell function, it is unclear which are dominant in the tumor microenvironment and thus may be viable therapeutic targets in cancer. Interleukin-35 (IL35), an Ebi3/Il12a heterodimer, is an inhibitory cytokine produced by Tregs that is a potent suppressor of T cell proliferation and function. Treg-derived IL35 can also promote the development of an induced regulatory T cell population that suppresses via IL35 (iTr35), which in turn can contribute to the suppressive intratumoral milieu. However, the physiological impact and mechanism of action of IL35 within the tumor microenvironment are largely unknown. In this competitive renewal application, we will determine how IL35 modulates the tumor microenvironment. AIM 1: Dissecting the function and fate of IL35+ Tregs in the tumor microenvironment. Using Ebi3 reporter, conditional and linage tracing mice, we will determine the physiological importance of IL35 production by Tregs in tumors and the role and fate of Ebi3+ versus Ebi3? Tregs. AIM 2: Determining the impact of IL35 on CD8+ T cells within the tumor microenvironment. Using Ebi3 conditional mice, we will determine the impact of Treg-derived IL35 on the function and fate of CD8+ T cells in tumors. This project will have a significant impact of our understanding of the physiological importance and mechanism of action of IL35 in the tumor microenvironment. Given that we discovered IL35 and have many unique tools to probe its function, we are in the best position to conduct this research.